Proteome profiling of hippocampus reveals the neuroprotective effect of mild hypothermia on global cerebral ischemia-reperfusion injury in rats

Abstract

Cerebral ischemia is one of the leading causes of disability and mortality worldwide. Blood reperfusion of ischemic cerebral tissue may cause cerebral ischemia-reperfusion (IR) injury. In this study, a rat model of global cerebral I/R injury was established via Pulsinelli's four-vessel occlusion (4-VO) method. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis were employed to examine the ipsilateral hippocampus proteome profiles of rats with/without MH (32 °C) treatment after IR injury. Totally 2 122 proteins were identified, among which 153 proteins were significantly changed associated with MH (n = 7 per group, fold change-1.5, p < 0.05). GO annotation of the differentially expressed proteins (DEPs) revealed that cellular oxidant detoxification, response to zinc ions, aging, oxygen transport, negative regulation of catalytic activity, response to hypoxia, regulation of protein phosphorylation, and cellular response to vascular endothelial growth factor stimulus were significantly enriched with MH treatment. The KEGG analysis indicated that metabolic pathways, thermogenesis, pathways of neurodegeneration, chemical carcinogenesis-reactive oxygen species, fluid shear stress and atherosclerosis, and protein processing in endoplasmic reticulum were significantly enriched with MH treatment. Importantly, changes in 16 DEPs were reversed by MH treatment. Among them, VCAM-1, S100A8, CaMKK2 and MKK7 were verified as potential markers associated with MH neuroprotection by Western blot analysis. This study is one of the first to investigate the neuroprotective effects of MH on the hippocampal proteome of experimental models of cerebral IR injury. These DEPs may be involved in the most fundamental molecular mechanisms of MH neuroprotection, and provide a scientific foundation for further promotion of reparative strategies in cerebral IR injury.

Figure 1

Protective effects of mild hypothermia on hippocampus neurological damage and cognitive dysfunction after cerebral ischemia-perfusion injury. (a) HE staining; (b) Nissl staining; the time (c) and path length (d) that rats spent to find the platform during 4 training days; The time (e) and distance (f) that rats spent in the target quadrant in the probe test; (g) the routes of rats in the probe test. Sham: Sham-operation group; IR: cerebral IR injury followed by normothermia (37 °C) group; IR + MH: cerebral IR injury followed by 4 h of MH (32 °C) group. Scale bars = 50 µm. Results are presented as the mean ± standard deviation (SD). *P < 0.05.

Figure 2

The differentially expressed proteins in hippocampus under cerebral IR injury followed by normothermia or MH. (a) Hierarchical cluster analysis; (b) the Venn diagram. Sham: Sham-operation group; IR: cerebral IR injury followed by normothermia (37 °C) group; IR + MH: cerebral IR injury followed by 4 h of MH (32 °C) group.

Figure 3

GO and KEGG enrichment analysis of the DEPs associated with mild hypothermia. (a) Biological process; (b) cellular component; (c) molecular function; (d) KEGG pathway (p value < 0.05).

Figure 4

STRING PPI network analysis of the DEPs associated with mild hypothermia. The number of nodes is 150, the average node degree is 2.36, and the average local clustering coefficient is 0.432 (p value < 0.01).

Figure 5

Validation of DEPs associated with mild hypothermia by Western blotting. (a) Representative blots visualizing the levels of VCAM-1, CaMKK2, MKK7 and S100A8 in the three groups. GAPDH was used as the loading control. (b) Densitometric values are expressed as the mean ± SD normalized to GAPDH (n = 4 per group). Sham: Sham-operation group; IR: cerebral IR injury followed by normothermia (37 °C) group; IR + MH: cerebral IR injury followed by 4 h of MH (32 °C) group. *p < 0.01; **p < 0.001.